Biology subjects

Nguyen, T. N.

Publications and source records attributed to Nguyen, T. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Isotype specific loss of HP1α but not of HP1β uncovers genomic regions that behave as HP1α-dependent common fragile sites

HP1 proteins are highly evolutionarily conserved chromatin-associated factors known to play essential roles in genome stability and nuclear organization. In mammals, three HP1 isoforms, HP1, HP1{beta} and HP1{gamma}, have been described, but their individual functions remain incompletely characterized. Here, we inactivated HP1 or HP1{beta} in different cell lines and quantified chromosomal breaks on metaphase spreads in the presence or absence of aphidicolin-induced replication stress. Loss of HP1, but not of HP1{beta}, led to a significant increase of chromosomal breaks on chromosome arms and within pericentromeric heterochromatin under these conditions. Mechanistically, loss of HP1 was associated with a reduction in replication fork velocity, suggesting that HP1 deficiency induces a replication stress that sensitizes specific genomic loci to replication perturbation. Consistent with this, HP1 loss was associated with a moderate but consistent increase in {gamma}H2AX and 53BP1 foci, an increased occurrence of DNA synthesis during mitosis, and enhanced recruitment of FANCD2, all recognized as hallmarks of common fragile site (CFS) expression. In addition, rescue experiments using a chromodomain mutant HP1 (V22M) unable to bind H3K9me3 indicated that HP1 protective function over these specific foci did not require its interaction with this histone mark. Altogether, these data indicate that, independently of its binding to H3K9me3, HP1 stabilizes specific genomic regions that behave as HP1-dependent fragile sites, at least in part by regulating replication fork progression, limiting mitotic DNA synthesis possibly by competing with FANCD2 for chromatin access at these regions.

cell biology↗

bioRxiv2025

mRNA-delivered neutralizing antibodies confer protection against SARS-CoV-2 variant in the lower and upper respiratory tract

Monoclonal antibodies (mAbs) have been developed as effective biological countermeasures against a range of human diseases. The high cost of antibody production and manufacturing limits its clinical application and widespread use. The mRNA-lipid nanoparticle (mRNA-LNP) is a versatile platform for development of vaccines and protein-replacement therapeutics. Since the COVID-19 pandemic, a number of neutralizing mAbs against SARS-CoV-2 have been identified with several being used clinically under emergency authorization. Herein, we report the design and generation of mRNA-LNPs expressing two SARS-CoV-2 neutralizing mAbs, 76E1 and LY1404, which respectively target the viral spike proteins fusion peptide (FP) epitope within the S2 subunit and the receptor-binding domain (RBD) within the S1 subunit. We show a single intramuscular administration of mRNA-LNPs results in efficient LY1404 and 76E1 mAb production in mice which is sustained for 7-14 days. Further, we evaluate the protective efficacy of mRNA-LNP formulations encoding the two antibodies in mouse and hamster models challenged with different SARS-CoV-2 viral strains. The data demonstrate that a single administration of mRNA-LNP encoding the more broadly neutralizing antibody 76E1 confers significant protection against the immune-evasive SARS-CoV-2 Omicron variant BQ.1 in both the upper and lower respiratory tract of the hamsters, indicating its potential impact on limiting both viral disease and viral acquisition. Together, our study expands the potential of the mRNA-LNP platform to deliver therapeutic antibodies for rapid prevention or treatment of pathogenic infections.

microbiology↗

bioRxiv2024

Whole-genome sequencing across space and time reveals impact of population decline and reduced gene flow in Florida Scrub-Jays

Whole-genome sequence data is proving to be highly informative about the past demography of free-living populations, and in the context of endangered species, it can provide a quantification of the genetic risk posed by reduced genetic diversity and inbreeding. Prior to 1920, the Florida scrub-jay (Aphelocoma coerulescens) had been widespread across Florida, but with the expansion of agriculture and human habitation, its population has declined by 95%, resulting in fragmentation into semi-isolated subpopulations. By sequencing 241 individuals sampled from five different regions and across two time points, this study quantifies a greater magnitude of loss of genetic diversity and greater levels of inbreeding in smaller and more isolated subpopulations. Consistent with population genetics theory, reduction in population size results in a dramatic loss of rare alleles, skewing the site frequency spectrum far from the expected equilibrium. Increased inbreeding in the smaller, more remote subpopulations is especially evident in the increased size and number of runs of homozygosity. The Florida scrub-jay displays limited dispersal, and habitat fragmentation has greatly reduced the magnitude of gene flow in the past 30 years, resulting in further decline of genetic diversity, especially in the peripheral populations. Analysis of these data is informative in guiding conservation efforts to retain genetic diversity and minimize the consequences of inbreeding in the Florida scrub-jay. HighlightsO_LIFive regional populations show distinct degrees of population isolation and decline. C_LIO_LIThere has been commensurate loss of genetic diversity, skewed site frequency spectra, reduced migration, and increased inbreeding (FROH). C_LIO_LIAs many state-wide populations decline, the smaller, more remote populations provide a glimpse into the future and a testbed for remediation approaches. C_LI

genomics↗

bioRxiv2024

Presynapses are mitophagy pit stops that prevent axon degeneration

Defects in neuronal mitophagy have been linked to neurodegenerative diseases including Parkinsons disease. However, despite the importance of mitophagy in neuronal homeostasis, the mechanistic basis for neurodegeneration when mitophagy is defective is unclear. Here, using human neurons, we discover that presynapses are mitophagy pit stops for damaged axonal mitochondria. We show that while mitochondrial damage and PINK1/Parkin activation events are distributed throughout axons, mitophagy initiation and autophagosome formation are restricted to presynapses, which we show contain the machineries required for mitophagy. Being the primary sites of axonal mitophagy, presynapses were vulnerable when PINK1/Parkin mitophagy was defective. We observed local cytochrome c release within presynapses from an accumulation of damaged mitochondria. This resulted in downstream degradative caspase activation, defining a mechanism for neurodegeneration. Pharmacological rescue of axon degeneration was achieved through synthetic upregulation of receptor mediated mitophagy with the clinically approved compound Roxadustat, revealing a potential therapeutic avenue for disease.

cell biology↗

bioRxiv2024

Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. While the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here, we demonstrate that transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, while FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with significant implications for therapeutic interventions.

cell biology↗

bioRxiv2024

A new high-quality genome assembly and annotation for the threatened Florida Scrub-Jay (Aphelocoma coerulescens)

The Florida Scrub-Jay (Aphelocoma coerulescens), a Federally Threatened, cooperatively-breeding bird, is an emerging model system in evolutionary biology and ecology. Extensive individual-based monitoring and genetic sampling for decades has yielded a wealth of data, allowing for the detailed study of social behavior, demography, and population genetics of this natural population. Here, we report a linkage map and a chromosome-level genome assembly and annotation for a female Florida Scrub-Jay made with long-read sequencing technology, chromatin conformation data, and the linkage map. We constructed a linkage map comprising 4,468 SNPs that had 34 linkage groups and a total sex-averaged autosomal genetic map length of 2446.78 cM. The new genome assembly is 1.32 Gb in length, consisting of 33 complete or near-complete autosomes and the sex chromosomes (ZW). This highly contiguous assembly has an N50 of 68 Mb and a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score of 97.1% with respect to the Aves database. The annotated gene set has a BUSCO transcriptome completeness score of 95.5% and 18,051 identified protein-coding genes, 92.2% of which have associated functional annotations. This new, high-quality genome assembly and linkage map of the Florida Scrub-Jay provides valuable tools for future research into the evolutionary dynamics of small, natural populations of conservation concern. ARTICLE SUMMARYWe present a new high-quality genome assembly and annotation for the Florida Scrub-Jay (Aphelocoma coerulescens), a Federally Threatened bird species. In comparison to other genome assemblies of this species, our assembly is the first to be made using long-read sequencing technology and is the first generated from a female individual. We also constructed the first linkage map for this species using a population pedigree. Our genome assembly is highly contiguous and is of similar quality to other bird genome assemblies.

genomics↗

bioRxiv2024

MICROMANIPULATION OF LIVE MICRODISSECTED TISSUES WITH A LOW-COST INTEGRATED ROBOTIC PLATFORM

The scarcity of human biopsies available for drug testing is a paramount challenge for developing new therapeutics, disease models, and personalized treatments. Microtechnologies that combine the microscale manipulation of tissues and fluids offer the exciting possibility of miniaturizing both disease models and drug testing workflows on scarce human biopsies. Unfortunately, these technologies presently require microfluidic devices or robotic dispensers that are not widely accessible. We have rapidly-prototyped an inexpensive platform based on an off-the-shelf robot that can microfluidically manipulate live microtissues into/out of culture plates without using complicated accessories such as microscopes or pneumatic controllers. The robot integrates complex functions with a simple, cost-effective and compact construction, allowing placement inside a tissue culture hood for sterile workflows. We demonstrated a proof-of-concept cancer drug evaluation workflow of potential clinical utility using patient tumor biopsies with multiple drugs on 384-well plates. Our user-friendly, low-cost platform promises to make drug testing of microtissues broadly accessible to pharmaceutical, clinical, and biological laboratories. TeaserA low-cost robot for handling microtissues and catalyzing their use in cancer drug evaluation and personalized oncology.

bioengineering↗

bioRxiv2024

Microdissected tumor cuboids: a microscale cancer model that retains a complex tumor microenvironment

To bridge the gap between bench and bedside, there is a need for more faithful models of human cancers that can recapitulate key features of the human tumor microenvironment (TME) and simultaneously facilitate large-scale drug tests. Our recently developed microdissection method optimizes the yield of large numbers of cuboidal microtissues (''cuboids '', ~(400 m)3 ) from a tumor biopsy. Here we demonstrate that cuboids from syngeneic mouse tumor models and human tumor biopsies retain a complex TME, making them amenable for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as cellular architecture, cytokine secretion, proteomics profiles, and response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong cytokine expression and drug responses, including to immunotherapy. Overall, our results suggest that cuboids could provide essential therapeutic information for personalized oncology applications and could help the development of TME-dependent therapeutics and cancer disease models, including for clinical trials.

cancer biology↗

bioRxiv2023

The T Cell Receptor beta Chain Repertoire of Tumor Infiltrating Lymphocytes Improves Neoantigen Prediction and Prioritization

In the realm of cancer immunotherapy, the meticulous selection of neoantigens plays a fundamental role in enhancing personalized treatments. Traditionally, this selection process has heavily relied on predicting the binding of peptides to human leukocyte antigens (pHLA). Nevertheless, this approach often overlooks the dynamic interaction between tumor cells and the immune system. In response to this limitation, we have developed an innovative prediction algorithm rooted in machine learning, integrating T cell receptor {beta} chain (TCR{beta}) profiling data from colorectal cancer (CRC) patients for a more precise neoantigen prioritization. TCR{beta} sequencing was conducted to profile the TCR repertoire of tumor-infiltrating lymphocytes (TILs) from 28 CRC patients. The data unveiled both intra-tumor and inter-patient heterogeneity in the TCR{beta} repertoires of CRC patients, likely resulting from the stochastic utilization of V and J segments in response to neoantigens. Our novel combined model integrates pHLA binding information with pHLA-TCR binding to prioritize neoantigens, resulting in heightened specificity and sensitivity compared to models using individual features alone. The efficacy of our proposed model was corroborated through ELISpot assays on long peptides, performed on four CRC patients. These assays demonstrated that neoantigen candidates prioritized by our combined model outperformed predictions made by the established tool NetMHCpan. This comprehensive assessment underscores the significance of integrating pHLA binding with pHLA-TCR binding analysis for more effective immunotherapeutic strategies.

cancer biology↗

bioRxiv2023

Genomic islands of speciation harbor genes underlying coloration differences in a pair of Neotropical seedeaters

Incomplete speciation can be leveraged to associate phenotypes with genotypes, thus providing insights into the traits relevant to the reproductive isolation of diverging taxa. We investigate the genetic underpinnings of the phenotypic differences between Sporophila plumbea and S. beltoni. S. beltoni has only recently been described based, most notably, on differences in bill coloration (yellow vs. black in S. plumbea). Both species are indistinguishable through mtDNA or reduced-representation genomic data, and even whole-genome sequencing revealed low genetic differentiation. Demographic reconstructions attribute this genetic homogeneity to gene flow, despite divergence in the order of millions of generations. We found a narrow hybrid zone in southern Brazil where genetically, yet not phenotypically, admixed individuals appear to be prevalent. Despite the overall low genetic differentiation, we identified three narrow peaks along the genome with highly differentiated SNPs. These regions harbor six genes, one of which is involved in pigmentation (EDN3) and is a candidate for controlling bill color. Within the outlier peaks we found signatures of resistance to gene flow, as expected for islands of speciation. Our study shows how genes related to coloration traits are likely involved in generating prezygotic isolation and establishing species boundaries early in speciation.

evolutionary biology↗

bioRxiv2023

Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD

Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PINK1/Parkin-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 adaptors NAP1 and SINTBAD restrict the initiation of OPTN-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of NDP52-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.

cell biology↗

bioRxiv2023

A single-dose intramuscular immunization of pigs with lipid-nanoparticle DNA vaccines based on the hemagglutinin antigen confers complete protection against challenge infection with the homologous influenza virus strain

Influenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as a nanocarrier to deliver DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. Intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only one out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines. ImportanceInfluenza A virus of swine (IAV-S) is a significant pathogen of swine. The virus also poses a great public health concern due to its zoonotic potential. Although whole-inactivated virus (WIV) vaccines are available to control IAV-S, their heterologous efficacy is limited due to the substantial genetic/antigenic variation of the viral genome. This study provides compelling evidence demonstrating that lipid nanoparticle encapsulated DNA (LNP-DNA) vaccines induce robust systemic and mucosal immunity and complete protection of pigs against challenge infection with the homologous IAV-S strain. Importantly, the LNP-DNA vaccine approach meets the regulatory requirements to serve as an adaptable platform that can be frequently updated to match the emergence of new IAV-S variants. Thus, this LNP-DNA vaccine strategy exhibits great potential in effectively mitigating the economic impact of IAV-S on the swine industry and the associated public health threat.

microbiology↗

bioRxiv2023

Interaction of PINK1 with nucleotides and kinetin

PINK1 is a ubiquitin kinase that accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinsons disease. Nucleotide analogues such as kinetin triphosphate (KTP) have been suggested to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinsons disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP. We establish a cryo-EM platform exploiting the previously observed dodecamer assembly of Pediculus humanus corporis (Ph) PINK1 to determine PINK1 structures bound to AMP-PNP and ADP, which reveal unexpected conformational changes in the kinase N-lobe to enable PINK1 to form a ubiquitin binding site. Strikingly, we find that KTP is unable to bind PhPINK1 or human (Hs) PINK1 due to a steric clash with the kinase gatekeeper residue. Mutation of the gatekeeper to Ala or Gly is required to enable PINK1 to bind and utilise KTP as a phosphate donor in ubiquitin phosphorylation and mitophagy. Indeed, HsPINK1 M318G can be used to conditionally uncouple PINK1 stabilisation and activity on mitochondria.

molecular biology↗

bioRxiv2023

Improvement in Neoantigen Prediction via Integration of RNA Sequencing Data for Variant Calling

Neoantigen-based immunotherapy has emerged as a promising strategy for improving the life expectancy of cancer patients. This therapeutic approach heavily relies on accurate identification of cancer mutations using DNA sequencing (DNAseq) data. However, current workflows tend to provide a large number of neoantigen candidates, of which only a limited number elicit efficient and immunogenic T-cell responses suitable for downstream clinical evaluation. To overcome this limitation and increase the number of high-quality immunogenic neoantigens, we propose integrating RNA sequencing (RNAseq) data into the mutation identification step in the neoantigen prediction workflow. In this study, we characterize the mutation profiles identified from DNAseq and/or RNAseq data in tumor tissues of 25 patients with colorectal cancer (CRC). We detected only 22.4% of variants shared between the two methods. In contrast, RNAseq-derived variants displayed unique features of affinity and immunogenicity. We further established that neoantigen candidates identified by RNAseq data significantly increased the number of highly immunogenic neoantigens (confirmed by ELISpot) that would otherwise be overlooked if relying solely on DNAseq data. In conclusion, this integrative approach holds great potential for improving the selection of neoantigens for personalized cancer immunotherapy, ultimately leading to enhanced treatment outcomes and improved survival rates for cancer patients.

immunology↗

bioRxiv2023

The Genetic Landscape of a Metabolic Interaction

Enzyme abundance, catalytic activity, and ultimately sequence are all shaped by the need of growing cells to maintain metabolic flux while minimizing accumulation of deleterious intermediates. While much prior work has explored the constraints on protein sequence and evolution induced by physical protein-protein interactions, the sequence-level constraints emerging from non-binding functional interactions in metabolism remain unclear. To quantify how variation in the activity of one enzyme constrains the biochemical parameters and sequence of another, we focused on dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), a pair of enzymes catalyzing consecutive reactions in folate metabolism. We used deep mutational scanning to quantify the growth rate effect of 2,696 DHFR single mutations in 3 TYMS backgrounds under conditions selected to emphasize biochemical epistasis. Our data are well-described by a relatively simple enzyme velocity to growth rate model that quantifies how metabolic context tunes enzyme mutational tolerance. Together our results reveal the structural distribution of epistasis in a metabolic enzyme and establish a foundation for the design of multi-enzyme systems.

biochemistry↗

bioRxiv2023

A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single- cell RNA-sequencing (scRNA-seq) dataset of [~]7 million cells profiled, and a spatially resolved transcriptomic dataset of [~]4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

neuroscience↗

bioRxiv2022

Temporal landscape of mitochondrial proteostasis governed by the UPRmt

Breakdown of mitochondrial proteostasis activates quality control pathways including the mitochondrial unfolded protein response (UPRmt) and PINK1/Parkin mitophagy. However, beyond the upregulation of chaperones and proteases, we have a limited understanding of how the UPRmt remodels and restores damaged mito-proteomes. Here, we have developed a functional proteomics framework, termed MitoPQ (Mitochondrial Proteostasis Quantification), to dissect the UPRmts role in maintaining proteostasis during stress. We discover essential roles for the UPRmt in both protecting and repairing proteostasis, with oxidative phosphorylation metabolism being a central target of the UPRmt. Transcriptome analyses together with MitoPQ reveal that UPRmt transcription factors drive independent signaling arms that act in concert to maintain proteostasis. Unidirectional interplay between the UPRmt and PINK1/Parkin mitophagy was found to promote oxidative phosphorylation recovery when the UPRmt failed. Collectively, this study defines the network of proteostasis mediated by the UPRmt and highlights the value of functional proteomics in decoding stressed proteomes.

cell biology↗

bioRxiv2022

Dual states of Bmi1-expressing intestinal stem cells drive epithelial development and tissue regeneration

Intestinal development, response to injury and disease states rely upon balanced stem cell proliferation. Historically, two subtypes of intestinal epithelial stem cells (ISCs)--slow-cycling/label-retaining, and actively-cycling/canonical Wnt-dependent--coordinate to drive proliferation and regulate epithelial renewal during adult tissue homeostasis and injury response. Recent studies focused on Bmi1-expressing cells revealed that differentiated Bmi1+ enteroendocrine cells could dedifferentiate towards a canonical Wnt-dependent stem cell state, calling into question the dogma that a dual stem cell axis regulates epithelial proliferation. Herein, we identify stem cell function in a Bmi1+ cell population in early murine intestinal development prior to the establishment of canonical Wnt-dependent, Lgr5-expressing ISCs. In-depth analyses of developmental Bmi1+ ISCs using lineage-tracing and single cell RNA-sequencing reveal their distinct identity and capacity to differentiate into Lgr5+ ISCs and other differentiated lineages. Further, during in utero development, the Bmi1+ ISCs initially exists in a highly proliferative state then transitions to a slow-cycling state, with the emergence of actively-cycling Lgr5+ ISCs. In adult tissue, Bmi1+ ISCs are a distinct population that re-express developmental gene and protein profiles, and a non-canonical Wnt signaling signature in response to injury and in human colorectal tumors. Further, developmental Bmi1+ ISCs are distinct from Lgr5+ ISCs and the previously identified differentiated Bmi1+ progenitor cells. Re-evaluation of an under-appreciated Bmi1+ ISC population with fundamental importance in intestinal development re-establishes the importance of the dynamic interplay between discrete ISC populations that are regulated by opposing Wnt signaling pathways. This finding opens opportunities and targetable pathways to augment regeneration or inhibit tumorigenesis.

developmental biology↗